Bottom Line:
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

ABSTRACTSalmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

Figure 2: NlpE-mediated activation of CpxA represses SPI-1 through CpxR. Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxR mutant carrying plasmid pCA-NlpE, grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. Expression (+) of NlpE from the T5-lac promoter of plasmid pCA-NlpE was induced by adding 50 μM IPTG at the beginning of the bacterial cultures.

Mentions:
Overproduction of the lipoprotein NlpE activates the kinase activity of CpxA and thus the CpxA-dependent phosphorylation of CpxR (Snyder et al., 1995; Hunke et al., 2012; Vogt and Raivio, 2012). Hence, to determine whether the CpxA-mediated activation of CpxR also represses the expression of the SPI-1 genes, we examined the effect of the overexpression of NlpE, from an IPTG-inducible promoter, on the protein secretion profiles of the WT S. Typhimurium strain and its derivative ΔcpxR mutant. Since Salmonella lacks NlpE, the E. coli K12 NlpE was used in these assays. As shown in Figure 2, the induction of the NlpE expression by the presence of IPTG decreased the secretion/expression of the SipA-D proteins in the WT strain but not in its derivative ΔcpxR mutant, indicating that the activation of CpxA represses the secretion/expression of SPI-1-encoded proteins through CpxR. To further confirm the regulatory role of CpxR on the SPI-1 genes, we determined the effect of its overexpression on the protein secretion profile of the WT S. Typhimurium strain, since the overexpression can bypass the need for phosphorylation of CpxR to regulate target genes (Macritchie et al., 2008; Acosta et al., 2015; Yun et al., 2015). The E. coli K12 CpxR is 97% identical to that of S. Typhimurium 14028s; thus, the plasmid pCA-CpxR from the ASKA library (Kitagawa et al., 2005), which expresses the E. coli K12 CpxR from an IPTG-inducible promoter, was used in these assays. As shown in Figure 3A, the overexpression of CpxR reduced the secretion/expression of the SipA-D proteins. Furthermore, the overexpression of CpxR repressed the expression of HilA-FLAG and InvF-FLAG in the WT S. Typhimurium strain (Figure 3B). In all, these results indicate that CpxA-mediated phosphorylation of CpxR or the overexpression of CpxR represses the expression of the SPI-1 genes.

Figure 2: NlpE-mediated activation of CpxA represses SPI-1 through CpxR. Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxR mutant carrying plasmid pCA-NlpE, grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. Expression (+) of NlpE from the T5-lac promoter of plasmid pCA-NlpE was induced by adding 50 μM IPTG at the beginning of the bacterial cultures.

Mentions:
Overproduction of the lipoprotein NlpE activates the kinase activity of CpxA and thus the CpxA-dependent phosphorylation of CpxR (Snyder et al., 1995; Hunke et al., 2012; Vogt and Raivio, 2012). Hence, to determine whether the CpxA-mediated activation of CpxR also represses the expression of the SPI-1 genes, we examined the effect of the overexpression of NlpE, from an IPTG-inducible promoter, on the protein secretion profiles of the WT S. Typhimurium strain and its derivative ΔcpxR mutant. Since Salmonella lacks NlpE, the E. coli K12 NlpE was used in these assays. As shown in Figure 2, the induction of the NlpE expression by the presence of IPTG decreased the secretion/expression of the SipA-D proteins in the WT strain but not in its derivative ΔcpxR mutant, indicating that the activation of CpxA represses the secretion/expression of SPI-1-encoded proteins through CpxR. To further confirm the regulatory role of CpxR on the SPI-1 genes, we determined the effect of its overexpression on the protein secretion profile of the WT S. Typhimurium strain, since the overexpression can bypass the need for phosphorylation of CpxR to regulate target genes (Macritchie et al., 2008; Acosta et al., 2015; Yun et al., 2015). The E. coli K12 CpxR is 97% identical to that of S. Typhimurium 14028s; thus, the plasmid pCA-CpxR from the ASKA library (Kitagawa et al., 2005), which expresses the E. coli K12 CpxR from an IPTG-inducible promoter, was used in these assays. As shown in Figure 3A, the overexpression of CpxR reduced the secretion/expression of the SipA-D proteins. Furthermore, the overexpression of CpxR repressed the expression of HilA-FLAG and InvF-FLAG in the WT S. Typhimurium strain (Figure 3B). In all, these results indicate that CpxA-mediated phosphorylation of CpxR or the overexpression of CpxR represses the expression of the SPI-1 genes.

Bottom Line:
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

ABSTRACTSalmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.